HU189706B - Improved process for the production of interferon - Google Patents

Abstract

An improvement to the process for the production of interferon by adding an inducer to lymphoblastoid cells susceptible to being induced to produce interferon is described wherein the cells are treated with an enhancing agent at, or following, induction. In a preferred aspect the cells are also pretreated, before induction, with a stimulator.

Description

Interferon is a protein that has non-specific antiviral activity by influencing cellular metabolic processes such as RNA and protein synthesis, at least in homologous cells. Interferons are of wide interest as potential antiviral and anticancer drugs. Several types of interferons are known, including alpha (leukocyte), beta (fibroblast) and gamma (immune) interferons. The present invention relates to the production of alpha interferons.

The primary sources of alpha interferons are human white blood cells, but they cannot provide the amount of interferon alpha needed for clinical purposes. Recently, methods have been described in which interferon alpha is produced by transformed human cells, namely human lymphoblastoid cells. (Advances im Experimental Medicine and Biology 1978, 110, 61-74, J. Clinical Microbiology

1978, 7, 44-51), and more recently large-scale production of interferon alpha has also been realized. (Antimicrobal Ágenst and Chemotherapy 1979 15,420-427, Texas Report Bioi. Med. 1981-82. 41, 175-178).

The steps of producing alpha interferon from human lymphoblastoid cells are as follows:

i) culturing the lymphoblastoid cells in a suitable medium, ii) inducing the cells to produce interferon by adding an inducer, iii) incubating the cells after induction, and iv) recovering the formed interferon.

Many techniques are known to increase the yield of interferon production by such a process.

For example, one of these methods is to treat the lymphoblastoid cells with a "pretreatment agent" or stimulator for some time before induction. Thus, European Patent 0 000 520 discloses a method for increasing the yield of interferon in which the pretreatment is carried out with a carboxylic acid or a salt thereof, namely sodium butyrate. Similar pretreatment procedures are known in the art using a variety of other compounds (Virology

1979, 99, 158-166, 0 008 391).

In such methods, the cells are usually exposed to the cells for a period of time, e.g. It is treated for 48 hours and the pretreatment agent is removed prior to induction.

Alternatively, an increase in interferon yield may be achieved by lowering the temperature of the cultured cells during or after induction (Proc. Nat. Acad. Sci. USA 1973, 70, 3909-3913, Japanese J. Microbiology 1974 18, 217- 222, Microbiol. Immunol., 1980, 24, 907-914, J. Gen. Virology 1981, 56, 163-174).

It was previously known in the literature that administration of the above-mentioned "stimulants" during or after induction adversely affects cellular interferon production. This is especially true of carboxylic acids such as butyric acid (Virology 1979 99, 158-166, Biochem. Biophys. Rés. Commun. 1981, 103, 806-812).

It has been found that administration of certain compounds (hereinafter referred to as "yield enhancing agents") during or shortly after induction of lymphoblastoid cells enhances interferon yield.

Accordingly, the present invention relates primarily to a method for producing the interferon, wherein inducible lymphoblastoid cells are stimulated by the addition of an inducer to produce interferon, characterized in that the cells are induced, or shortly thereafter, with a yield-enhancing agent of the invention.

For the production of interferon, lymphoblastoid cells are selected according to need, for example, human cells for the treatment of human interferon are usually used. Namalwa cells or other suitable lymphoblastoid cell lines may be advantageously used for this purpose. Cultured lymphoblastoid cell lines that can be propagated in culture can be readily derived from cultures of human peripheral blood leukocytes by well-known methods. (Hope, J. H., Home, J. K., Scott, W. "Int. J. Cancer, 1978, 3, 857866, Hope, J. H., Home," K. Scott, W., Int. J. Cancer 1969, 4, 255-260, Chang, RS, Goiden, HD, Natura 1971, 234, 359-360) Accordingly, leukocytes can be obtained from healthy or diseased individuals, either "spontaneously derived when already infected with Epstein-Barr virus (EBV). , or derived from cultures of leukocytes not infected with EBV, e.g. leukocytes obtained from umbilical cord blood to which EBV is added.

Lymphoblastoid cell lines can be easily obtained from cells of patients with Burkitt's lymphoma since they are already infected with EBV. A particular cell line, Namalwa, was derived by Professor G. Klein in Stockholm (Nyormoi, O., Klein, G., Adams, A., Dombos, L., Int. J. Cancer 1973, 12, 396-408). girl cells. Cells in this cell line have been found to be able to produce a large amount of interferon when properly stimulated (Strander, H., Mogesen,

K. E., Cantell, K., J. Clin. Microbiol. 1975, 1, 116-117, Christophinis, G.J., Seli, C.M. and Finter, N.B., J.Gen. Virol. 1981, 52, 169-171). A subculture of this cell line was obtained in January 1975 from Dr. Ion Gresser (Villejuif, France). The cell line was then acclimated to growth on RMPI 1640 medium supplemented with 10% fetal bovine serum.

The cells were acclimated by Wellcome Research Laboratories researchers for growth on the same medium supplemented with 5-7% serum from 6-8 months old calves, and the culture was inoculated twice or three times a week for 18 months. From these cells, a Basic Bank, now known as Namalwa (WRL) was created with a number of ampoules stored in liquid nitrogen. CRL No. 1432. In the following examples, Namalwa (WRL cells) are included, but the invention is equally applicable to other Namalwa subcultures as well as other suitable lymphoblast sphid cells.

The term "enhancing agent" refers to any compound that, when administered shortly or shortly after administration, significantly increases the yield of interferon compared to that obtained in the absence of the agent.

A large number of different compounds have proven to be effective in yielding grains. Many of these

189,706 were previously mentioned as "stimulants" for pre-induction of cells. However, the most preferred stimulants, alkanoic acids (especially butyric acid) and their salts, as well as steroids (such as dexamethasone), are non-yielding agents and even reduce interferon yields, if present during or after induction.

Each of the compounds having a yield-enhancing effect also has the effect of inducing differentiation of Friend cells (although not all Friend cells have a yield-enhancing effect). (Chemically Induced Rat Erythrolukemia Differentiation, Reuben, R.C., et al., Biochemica et Biophysica Acta, 1980, 605,325-346).

Effective yield increasing agents are the following: N-Acetyl-p-aminophenol,

Acetamide and N- (C 1-6 alkyl and dialkyl) acetamides optionally substituted with a hydroxy group (e.g. N-methylacetamide,

Ν, Ν-dimethylacetamide, N-ethylacetamide, N, N-diethylacetamide, N-butylacetamide, N-acetyl-6-hydroxyhexylamine),

Acetanilide and lower alkoxyacetanilides (eg p-ethoxyacetanilide),

C 4 -C 8 alkylene bis-acetamides (such as hexamethylene bis-acetamide, pentamethylene bis-acetamide, heptamethylene bis-acetamide),

Ν, Ν-Diemtil hexanamide,

Dimethylsulfoxide,

Lower alkanoic amides (eg propionamide, butyramide),

Lower alkyl benzamides (e.g. N-methylbenzamide)

Lower alkyl-2-imidazolidinones (e.g., 1,3-dimethyl-2-imidazolidinone),

Lower alkyl 2-pyrrolidinones (e.g. 1-methyl-2-pyrrolidinone,

ethyl-2-pyrrolidinone,

2-Piperidone and 1-lower alkyl-2-piperidones (e.g., 1-methyl-2-piperidone),

Piri din N-oxide as well

Urea and thiourea derivatives (e.g., tetramethylthiourea, lower alkyl ureas such as 1-methylurea, 1-ethylurea, 1-propylurea, 1,1-dimethylurea, , 3-dimethylurea, 1,3-diethylurea).

Particularly preferred compounds are: N-acetyl-p-aminophenol;

N, N-dimethylacetamide,

13-dimethyl-2-imidazolidinone, dimethyl sulfoxide, p-ethoxyacetanilide, hexamethylene bis-acetamide,

N-methylacetamide,

1-methylpiperidone, 1-methyl-2-pyrrolidone, and tetramethylurea. "

The term "lower alkyl" as used herein means an alkyl group having 1 to 4 heteroatoms. In order to produce interferon, the selected cells must first be cultured under conditions which are known in the literature to be of a particular cell type or strain. For example, human lymphoblastoid cells, such as the Namalwa cell line, grow well with serum, typically 5-10% v / v calf or horse serum, in supplemented RPMI 1640 medium (MOORE, G.E., et al.,

J. Amer. Med. Assoc. 1967, 199, 519-524).

During interferon production with lymphoblastoid cells (e.g., Namalwa cells), the cells are first cultured until a sufficient concentration of suspension is obtained.

The concentrations, conditions, etc., required for interferon production are well known and appreciated by those skilled in the art.

During this step, the yield enhancing agent is added. It is best to add the agent during induction (i.e., when an inducing agent such as Sendai virus is added to the culture to induce interferon production), i.e., the yield enhancer may already be present in the cell suspension medium immediately before or immediately after induction. to be added to the cell suspension. However, the beneficial effect of the present invention is also seen when the yield-enhancing agent is added a little later, but within two hours after induction.

The amount of yield-enhancing agent used is limited by the toxicity of the cell line, but such amount should be such as to increase the yield of interferon. The optimum concentration obtained as a result of the toxic and yield enhancing effect is different for each yield enhancing agent, but is generally 0.1 to 500 mM, preferably 1 to 200 mM, most preferably 5 to 50 mM.

Induction and subsequent incubation may be effected in any known manner for the production of interferon. For examples the cells resuspended in medium RPMI 1640 - no serum or supplemented with 5 percent serum volume) and 0.25 6xlO ^these cells / ml, preferably from 0.5 to 3x10 cells / ml concentration limits. A suitable inducer, such as viruses such as Sendai virus (Johnston, MDJ Gene. Virol. 1981 56, 175-184), is then added to the cell suspension at 5 and 200 Units of haemagglutination per ml (HAU / ml), preferably at 20 and 50. At a final concentration of HAU / ml. After thorough mixing, the cell suspension is incubated at 34-37 ° C for 12-48 hours. For example, when incubated at 35 ° C overnight, the resulting culture of interferon is transferred from the cells to the culture medium. The cells are then removed, for example by centrifugation, to obtain a supernatant containing crude interferon, which can be purified, if necessary, in a known manner. (Definition of hemagglutination unit: Granóff, A. et al., Studies on the Interaction of Large and Small Heamagglutinating Components of New-Cartel Disease Viras with Red Blood Cells, 1954, 72, 329-339). The yield increase when using a yield enhancer is very significant and may reach up to five times the value in the absence of the agent.

The mechanism of action of the yield enhancing agent is not yet fully elucidated. However, evidence suggests that the rate of interferon production increases for a longer time than in the absence of the drug. This phenomenon is due to the fact that interferon m-RNA can be produced over a longer period of time, which is a true superinduction. Accordingly, the present invention also relates to a method of producing interferon, wherein inducible lirrh-oblateoid cells are stimulated by the addition of an inducer to produce interferon, characterized in that the rate of interferon production following the induction is significantly prolonged.

It takes 189,706 more minutes, by the way.

"By the way, the term" interferon is produced by a method of producing interferon with lymphoblastoid cells in a manner known in the art, but without affecting in any way the duration of the increase in the rate of interferon formation,

Prolongation of the growth time interval of the interferon formation rate can be achieved by increasing the interferon mRNA transcription period, i.e., by superinduction.

This increase in duration is conveniently accomplished using the yield enhancing agents and conditions of the present invention. The invention thus also relates to a method by which the period of interferon production can be prolonged as compared to conventional interferon production by known methods.

The present invention can very advantageously be combined with known methods of increasing the yield of interferon production by lymphoblastoid cell lines, so that both temperature reduction and pre-treatment with a stimulator prior to induction can be combined with the combination to provide better yield of interferon than individual methods alone. -Separate. In particular, pretreatment of cells with a stimulator prior to induction (European Patent Nos. 0, 000, 520 and 0, 008, 391, both of which are incorporated herein by reference) has proven to be advantageous.

When a stimulant can be used as a yield enhancer, two functions of the same compound can be utilized. Preferably, however, the pretreatment is carried out according to the method of European Patent 0 000 520, i.e. with an alkanoic acid or a salt thereof, namely butyric acid or sodium butyrate.

Accordingly, the present invention also relates to a method of producing interferon by providing inducer to lymphoblastoid cells that are stimulated to produce interferon and which have previously been incubated in a medium containing a sufficient but non-toxic amount of stimulant and shortly thereafter during the induction process.

The present invention also relates to a method for producing interferon, wherein an inducer is added to lymphoblastoid cells that are stimulated to produce interferon and have previously been incubated in a medium containing a sufficient but non-toxic amount of stimulant and wherein the rate of interferon production extended.

The following examples illustrate the invention without, however, limiting its scope.

Example 1

Effect of yield enhancing agents on interferon production

A series of experiments were performed in which Namalwa / WRL cells were cultured on PRMI 1640 medium containing 5% adult bovine serum, polymyxin, neomycin and pH-adjusted sodium bicarbonate. When the cell concentration reached 2x10 * cells / ml, a sample was taken and diluted twice with said medium. The cell suspension was then added with 1 mM sodium butyrate and incubated with stirring at 37 ° C for 48 hours. After centrifugation at 800 g after the incubation period, cells were harvested and resuspended in RPMI 1640 medium containing 2% adult bovine serum at a concentration of 3x10 * cells / ml. To the cell suspension, 10 HAU / ml Sendai virus was added to induce interferon formation, and the induced cells were plated into 25 cm ³ plastic flasks every 10 ml. Each flask was treated with various yield enhancing agents as shown in Table 1. Two flasks were left for untreated control. After 24 hours, the cells were removed by centrifugation (800 g, 5 minutes), and the clear supernatant containing interferon was acidified to pH 2, stored at 4 ° C for 24 hours, and the interferon content was determined. The increase in interferon titer relative to the standard control (untreated) was calculated for each yield enhancing agent. The

results in Table I They included: For the induced culture The yield- A control given yield enhancing agent final concentration of the enhancer (mM) relative relative yield- growth acetamide 20 2 acetanilid 2 2 N-acetyl-6-hydroxyhexyl-alanine 5 3 N-butyl-acetamide 5 5 butyramide 10 3 N, N-diethylacetamide 10 4 1,3-diethyl-urea 5 3 Ν, Ν-dimethylacetamide 5 2 N, N-dimethylhexane amide 3 2 1,3-dimethyl-2-imidazolidine 2 3 EÍmetil sulfoxide 140 4 1,1-dimethylurea 10 2 1,3-dimethylurea 5 3 p-ethoxy-acetanilide 2 2 N-ethyl-acetamide 10 3 N-ethyl pyrrolidinone 5 3 Ethyl-urea 20 3 Hexamethylene bisacetamide 5 7 p-hydroxy acetanilide 5 3 N-methylacetamide 10 2 N-methylbenzamide 4 3 N-methyl-2-piperidone 5 3 N-methyl-2-pyrrolidinone 10 4 Methylurea 50 2 2-piperidone 25 2 propionamide 20 3 Propyl-urea 10 3 Pyridine N-oxide 50 4 Tetramethyl thiourea 5 4 T etram ethyl urea 8.4 6

Example 2

Effect of yield enhancing agents on interferon production with or without butyrate pretreatment

The Namalwa / WRL cell suspension prepared according to Example 1 was diluted to 1x10 * cells / ml with fresh medium. The culture was divided into two equal portions and one mM sodium butyrate was added. Both cultures were incubated for 48 hours with stirring and then induced with Sendai virus as in Example 1. The Induced

189,706 cell suspension suspensions were supplemented with the yield enhancers listed in Table 2. Interferon was isolated and assayed as in Example 1.

The Namalwa At induction The yield- interferon cells pre- dosed ho- enhancer yield a his treatment zamnövelő- concentration control of during the course agent • of the company compared to

(mM) sodium butyrate concentrate

cation (mM) 0 control _ 1 0 (No additions) acetanilid 2 3 0 Ν, Ν-dimethyl- acetamide 5 3 0 dimethylsulfoxide 140 2 0 N-methyl- benzamide 4 3 0 tetramethyl- -urea 8.4 6 1 control (No additions) 1 1 acetanilid 2 2 1 N, N-dimethyl- acetamide 5 2 1 dimethylsulfoxide 140 2 1 N-methyl- benzamide 4 3 1 tetramethyl- -urea 8.4 3

Example 3

Use of dimethyl sulfoxide as both stimulant and yield enhancer

The Namalwa / WRL cell suspension prepared in Example 1 was diluted to 1x10 * cells / ml with fresh medium. The culture was divided into two equal portions and 140 mM dimethylsulfoxide was added to one. Both cultures were incubated for 48 hours with stirring, then centrifuged at 800 g and cells were suspended in fresh medium (3x10 * cells / ml). Interferon induction was performed as in Example 1 and 140-140 mM dimethyl sulfoxide was added to one of the pre-treated and incubated and one of the untreated induced cell suspensions. Interferon was recovered and assayed after 24 hours as described in Example 1.

Namalwa cell At the induction interferon pretreatment added di- titer (10g ₁₀ applied di- methyl sulfoxide reference unit methyl sulfoxide concentration (Ml) concentration (MM) (MM) 0 0 3.55 140 0 4.0 0 140 3.9 140 140 43

Example 4

Kinetics of interferon formation in the presence or absence of a yield enhancing agent, with or without butyrate pretreatment

The Namalwa / WRL cell suspension prepared according to Example 1 was diluted to 1x10 * cells / ml with fresh medium. The culture was divided into two equal portions and one mM sodium butyrate was added. Both cultures were incubated for 48 hours with stirring, then centrifuged at 800 g and cells were suspended in fresh medium (3x10 cells / ml). Interferon induction was performed as in Example 1 and 8.4-8.4 mM tetramethylurea was added to half of the pre-treated and induced and untreated induced cell suspensions. The other half of the cultures did not receive tetramethylurea. Culture samples were centrifuged at 800 g every two hours for the four groups thus obtained (pre-treated, no-boost, no pre-boosted). The medium was removed and the cell mass was thoroughly drained. Cells were harvested to original volume in fresh medium and tetramethylurea was added to the cell suspension, if present prior to centrifugation. After a further two hours incubation, interferon was recovered and assayed according to Example 1.

Interferon titer (10g _lo reference unit / ml) Induction Untreated Namalwa Pretreated Namalwa dose and cells (ImM sodium terferonium butylate)

winning rocks Yield- 8.4 mM Yield- 8.4 mM it has passed raising agent tetrametU- raising agent tetramefil- Time (hours) néikül -urea- song without -urea- song 0-2 0.75 0.75 1.05 1.05 2-4 0.75 0.75 2.6 2.7 4-6 1.8 1.7 4.05 4.2 6-8 2.55 2.9 4.25 4.65 8-10 2.7 3.5 4.1 4.9 10-12 2.8 3.75 3.55 4.65 12-14 2.6 3.75 3.55 4.5 14-16 2.6 3.7 3.25 4.1 16 ^ 18 2.45 3.55 3.15 3.95

Example 5

The effect of butyrate administered during Induction with or without butyrate pretreatment.

The Namalwa / WRL cell suspension prepared according to Example 1 was centrifuged at 800 g, the cells were resuspended in fresh medium (3x10 cells / ml) and the interferon induction was performed as described in Example 1. Immediately after induction with Sendal virus, sodium butyrate was added to the cells at the concentrations shown in the table below. Interferon was recovered and assayed after 24 hours as described in Example 1.

189,706

Concentration of sodium butyrate added to the induced culture (mM)

0.1

0.2

0.5

Interferon titer (hanging reference unit (ml) 438)

432

433 4.27 4.17 4.14 3.93 3.83

Claims (10)

An improved process for the production of interferon comprising adding an inducing agent and optionally a stimulant to lymphoblastoid cells inducible for the production of interferon, characterized in that the cells are treated with a yield enhancing agent at the time of induction or immediately thereafter, N-acetyl-p-aminophenol, acetamide , optionally substituted with a hydroxy group, N- (C 1 -C 6 alkyl and dialkyl) acetamides, acetanilide, C 1 -C 4 alkoxyacetanilides, C 4 -C 8 alkylene bis-aleaamides, N, N-dimethylhexanamide, dimethyl sulfoxide , C 1 -C 6 alkanoic acid amides, C 1 -C 4 alkyl benzamides, mono- or di (C 1 -C 4 alkyl) -2-imidazolidines, 1- (C 1 -C 4 alkyl) -2-pyrrolidines, 2-piperidone , Using 1- (C 1 -C 4 alkyl) -2-pyridones, pyridine N-oxide or one to four (C 14 alkyl) substituted urea or thiourea. The process according to claim 1, wherein the yield-increasing agent is N-acetyl-5-p-aminophenol, Ν, Ν-dimethylacetamide, 13-dimethyl-2-imidazolidinone, dimethylsulfoxide, p-ethoxy, acetanilide, hexamethylene bis-acetamide, 1-methylpiperidinone or tetramethylurea. 3. A process according to any one of claims 1 to 4, characterized in that the yield is increased 10 agents are used in an amount of 0.1 to 500 mmol. The process according to claim 3, wherein the yield increasing agent is used in an amount of from 1 to 200 mmol. 5. The method of claim 3 wherein je pegylated floor 11, the concentration of enhancing agent is 5 to 50 mmol is used ^'0 amount. 6. A process according to any one of claims 1 to 4, characterized in that the yield enhancing agent is applied at the same time as the induction. 7. The el2Q process according to any one of claims 1 to 3, characterized in that the yield increasing agent is added within 2 hours after the addition of the inducer. 8. The method of any of claims 7, wherein the cells are pretreated with a stimulator prior to induction. 25 9. The process according to claim 8, wherein the stimulant is a straight-chain alkanoyl carboxylic acid having 1 to 6 carbon atoms or a salt thereof. The process of claim 9, wherein the alkanoyl is butyric acid as the carboxylic acid 30 are used.